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Full text of "Prostaglandin E2 Prevents Ovariectomy-Induced Cancellous Bone Loss in Rats"

NASA-CR-202468 



0169-6009/92/S05.00 C 1992 Elsevier Science Publishers B.V. All rights reserved. 



Bone and Mineral. 19 (1992) 45-62 ' 45 



BAM 00471 



Prostaglandin E 2 prevents ovariectomy-induced 
cancellous bone loss in rats 



Hua Zhu Ke ab , Mei Li a and Webster S.S. Jee a 

a Division of Radiobiology , University of Utah School of Medicine, Salt Lake City. UT, USA 
ti Bone Biology Laboratory. Zhanjiang Medical College, Guangdong Province, People's Republic of China 

(Received 3 March 1992) 
(Accepted 21 May 1992) 



Summary 

The object of this study was to determine whether prostaglandin E 2 (PGE 2 ) can prevent ovariectomy- 
induced cancellous bone loss. Thirty-five 3-month-old female Sprague-Dawley rats were divided into two 
groups. The rats in the first group were ovariectomized (OVX) while the others received sham operation 
(sham-OVX). The OVX group was further divided into three treatment groups. The daily doses for the 
three groups were 0, 1 and 6 mg PGE 2 /kg for 90 days. Bone histomorphometric analyses were performed 
on double-fluorescent-labeled undecalcified proximal tibial metaphysis (PTM). We confirmed that OVX 
induces massive cancellous bone loss ( — 80%) and a higher bone turnover (+ 143%). The new findings 
from the present study demonstrate that bone loss due to ovarian hormone deficiency can be prevented by 
a low-dose (1 mg) daily administration of PGE 2 . Furthermore, a higher-dose (6 mg) daily administration 
of PGE2 not only prevents bone loss but also adds extra bone to the proximal tibial metaphyses. PGE 2 at 
the 1-mg dose level significantly increased trabecular bone area, trabecular width, trabecular node density, 
density of node to node, ratio of node to free end, and thus significantly decreased trabecular separation 
from OVX controls. At this dose level, these same parameters did not differ significantly from sham-OVX 
controls. However, at the 6-mg dose level PGE 2 , there were significant increases in trabecular bone area, 
trabecular width, trabecular node density, density of node to node, and ratio of node to free end, while 
there was significant decrease in trabecular separation from both OVX and sham-operated controls. The 
changes in indices of trabecular bone microanatomical structure indicated that PGE 2 prevented bone loss 
as well as the disconnection of existing trabeculae. In summary, PGE 2 administration to OVX rats 
decreased bone turnover and increased bone formation parameters resulting in a positive bone balance 
that prevented bone loss (in both lower and higher doses) and added extra bone to metaphyses of OVX 
rats (in higher dose). These findings support the strategy of the use of bone stimulation agents in the 
prevention of estrogen depletion bone loss (postmenopausal osteoporosis). 



Key words: Prostaglandin E 2 ; Prevention; Ovariectomy; Cancellous bone; Architecture; Bone remodeling 



Correspondence to: Webster S.S. Jee, Ph.D., Building 586, Division of Radiobiology, University of 
Utah, Salt Lake City, UT 841 12, USA. 



46 

Introduction 

The positive bone balance (anabolic) effects of exogenous prostaglandin E 2 
(PGE 2 ) on bone are well known: daily administration of PGE 2 can increase 
bone mass in both animals [1-12] and man [13-15]. 

Previously, we reported that exogenous PGE 2 not only increased bone mass in 
intact male and female rats by activating bone modeling and remodeling in favor 
of bone formation [16-22], but also restored bone to osteopenic ovariectomized 
(OVX) rats [16-18]. In light of these facts, we postulated that PGE 2 will prevent 
ovariectomy-induced bone loss if given immediately following ovariectomy. The 
present study tested this postulate. It used a 90-day experimental period to allow 
both bone loss in OVX rats and bone gain in PGE 2 -treated rats to reach a new 
steady state [19,23]. This period equals at least two to three bone remodeling 
cycles [19,24]. 



Materials and Methods 

Thirty-five 3-months-old virgin female Sprague-Dawley rats, weighing approxi- 
mately 255 g (Charles River Laboratory, Inc., Portage, MI) were acclimated to 
local vivarium conditions (24 C C and 12 h/12 h light-dark cycle) for 7 days. During 
the experimental period, each animal was housed in a separate plastic module (21 
x 32 x 20 cm) and allowed free access to water and a pelleted (commercial) 
natural-product diet (Rodent Laboratory Chow 5001, Ralston-Purina Co., St. 
Louis, MO), which contained 1.46% of calcium, 0.99% of phosphorus and 4.96 
IU/g of Vit. D 3 . The rats were divided into five groups (6-9 rats per group). The 
first group (six rats) was sacrificed at day for baseline or basal controls. Group 2 
(six rats) was sham-OVX and treated simultaneously with a vehicle injection for 
90 days. Groups 3-5 were OVX and treated simultaneously with (six rats), 1 
(eight rats) and 6 (nine rats) mg PGE 2 /kg/day, respectively, for 90 days. 

All rats, except those killed at day 0, received daily injections of 1 ml/kg PGE 2 
or a vehicle solution. Powdered PGE 2 (The Upjohn Co., Kalamazoo, MI) was 
prepared as previously [19]. Groups 2 (sham-OVX controls) and 3 (OVX con- 
trols) received a vehicle (20% ethanol) while group 4 (OVX + 1 mg/kg/day) 
received 1 mg/ml, and group 5 (OVX + 6 mg/kg/day) received 6 mg/'ml daily 
subcutaneously on the back [19]. 

All rats received a subcutaneous injection of 25 mg/kg of tetracycline (Acho- 
mycin-tetracycline hydrochloride; Lederle Laboratory, Pearl River, NY) on the 
14th and 13th day and 10 mg/kg of calcein (Sigma Chemical Co., St. Louis, MO) 
on the 4th and 3rd day before sacrifice. 

At autopsy, all rats were anesthetized by intraperitoneally injecting a mixed 
solution of 50 mg/kg of ketamine hydrochloride (Veterinary Products, Bristol 
Laboratories, Div. of Bristol-Myers Co., Syracuse, NY) and 10 mg/kg of xylazine 
(Mobay Corporation, Animal Health Division, Shawnee, KS) at 1 ml/kg body 
weight. The rats were exsanguinated by heart puncture and the serum was stored 



47 

frozen, but was not analyzed because there were numerous publications on the 
subject [11,16,19,25,26]. The lungs, liver, adrenal glands, spleen, kidneys and 
thymus were removed and weighed. These organ weights were normalized to 
body weight as follows: mean body weight of all rats divided by body weight of 
each rat times organ weight. 

The left tibiae were removed and stored in 70% ethanol for the measurement of 
tibial length. The length of left tibia was measured using a Boley Millimeter 
Caliper Gauge (The L.S. Starrett Co., Athol, MA). 

The right tibia was removed, dissected and cut into three equal parts. The 
proximal third was processed to 100 /im and then microradiographed. Thereafter 
sections were further ground to a thickness of 20 /zm and coverslipped for 
morphometric measurements [16,19-21,25-28]. 

Using a Video Image Analysis System and KSS Image Analysis, we measured 
total tissue area, trabecular bone area and perimeter to calculate percent 
trabecular bone area, trabecular width, number and separation [29] in micro- 
radiographs of proximal tibial metaphyseal area between 1 and 4 mm distal to the 
growth plate-metaphyseal junction [30], We also measured the microanatomic 
trabecular bone structural indices which were previously defined by Garrahan et 
al. [3 1-34]. The indices included: number of nodes, node to node, node to free end, 
cortex to node, free end to free end and cortex to free end. These numbers were 
normalized to total tissue area and trabecular bone area in order to calculate their 
tissue- and bone-based densities. These indices provided data on the intercon- 
nectedness of the trabecular bone structure and on the number of structural 
elements. Node to node and cortex to node indicated interconnectedness. while 
free end to free end, node to free end and cortex to free end represented a lack of 
interconnectedness. When the trabeculae or struts were connected, the node to 
free end ratio was high, and low when the structures were broken [31-34]. 

A digitizing image analysis system (DIAS) was used for the static and dynamic 
histomorphometric measurements of the proximal tibial metaphyseal area be- 
tween 1 and 4 mm distal to the growth plate-metaphyseal junction (same as in 
microradiographs). The parameters included total tissue area, trabecular bone 
area and perimeter, eroded perimeter, osteoid perimeter, and trabecular wall 
width, single-labeled perimeter, double-labeled perimeter, and interlabel width 
(at the trabecular surface and the growth plate-metaphyseal junction region). 
These parameters were used to calculate percent trabecular bone area, trabecular 
width, number and separation [30], as well as percent osteoid perimeter, percent 
eroded perimeter, percent labeled perimeter, mineral apposition rate, bone for- 
mation rate-bone area and tissue area referent, formation period, resorption 
period, remodeling period, quiescent period, activation frequency and longitudi- 
nal growth rate [16,17,19-21,25-29,35]. We included an additional observation 
and measurement, the frequency and the amount of diffusely labeled trabecular 
bone, which has been defined previously [18]. Furthermore, we confirmed the 
presence of woven bone by the irregular orientation of osteocytes and collagen 
bundles by bright field and polarized light microscopes [36]. This parameter 
represents woven bone formed at 14, 13 and 4, 3 days before sacrifice when 



48 



fluorescent markers were injected, and it underestimates the total amount of 
woven bone [18]. We estimated the mean accumulated longitudinal growth as 
follows: ((LGR basal + LGR rmal )/2) x 90 days (newly generated metaphysis 
during the study period). 

Statistical differences between basal controls and other groups were evaluated 
using the two-tail Student Mest. The statistical differences between age-matched 
control and treatment groups were evaluated using ANOVA with Dunnett's r-test 
[37]. 



Results 

Effects on body weight 

Figure 1 shows changes in body weight with time. Compared to sham controls, 
the body weight of OVX control rats began to increase 20 days post OVX and 
continued increasing until 60 days post OVX, then plateaued thereafter. This 
finding is consistent with findings by others [38^40,46,49]. In OVX rats treated 
with 1 mg PGE 2 /kg/day, body weight increased by 12 to 18% from sham controls 
but was unchanged from OVX controls, while in OVX rats treated with 6 mg/kg/ 
day, body weight increased compared to sham controls (8 to 10%), but decreased 
compared to OVX controls (6 to 12%). 

Effects on soft tissue weights 

Figure 2 shows the changes in soft tissue weights after normalization to body 
weight. Compared to basal controls, spleen weight decreased (8%) in sham 
controls. Compared to sham controls, OVX control rats exhibited decreased 
liver and lung weights (8 and 9%), and increased spleen and thymus weights 
(14 and 55%). In PGE 2 -treated OVX rats (1 and 6 mg/kg/day), there was an 



Body weight (g) 

340 



320" 



.-+ 1— --f h OVX Controls 



OVX+ 1 mg 




OVX+ 6 mg 



Sham Controls 



90 Days 



Fig. 1. Time course of body weight changes in sham-operated controls, OVX controls and PGE 2 -treated 
OVX rats. "/><0.05 vs. sham-operated controls; *P<0 .05 vs. OVX controls; ><0.05 vs. OVX + 1 mg. 



49 



A. Liver weight (g) 

1ST 



13 



















T 


<§> 
— + 


# 




















S'd6< 

IB 






■a 

03 


| 


to 


1 + 
i> 

|o 



# * 



C. Kidneys weight (g) 

2.5 -I 



2.3- 



2.1" 



E. Adrenal glands weight (g) 



0.15 1 










0.13" 




0.11 - 




0.09- 


T T 




T 






0.07- 










n n< - 




3 


§ 


2*1 



B. Lungs weight (g) 





D. Spleen weight (g) 

0.8" 





F. Thymus weight (g) 



0.4" 




@> 


t 


0.3" 




T 






1 


0.2- 














^ 


0.1 - 










nn- 




I 


§ 

-G 
CO 





Fig. 2. Organ weight changes in basal controls, sham-operated controls, OVX controls and PGE 2 -treated 
OVX rats. Y error bar represents standard deviation. f P<0.05 vs. basal controls; "/><(). 05 vs. sham- 
operated controls; *P<0.05 vs. OVX controls; Y<0.05 vs. OVX + 1 mg. 



increase in weight of liver (12 and 55%), lungs (13 and 31%), adrenal glands (27 
and 53%), and a decrease in weight of thymus (26 and 54%) compared to OVX 
controls. In the OVX rats treated with 1 mg PGE 2 /kg/day, kidney weight 
decreased (11%) while adrenal gland weight increased (15%) when compared 
to those of sham controls. In the OVX rats treated with 6 mg PGE 2 /kg/day, 



50 



A. Tibial length (mm) 

411 

40 H 




Fig. 3. Effects on tibial length in basal controls, sham-operated controls, ovariectomized controls and 

PGEi-treated ovariectomized rats. Y error bar represents standard deviation. *P<0.05 vs. basal controls; 

'"P<0.05 vs. sham-operated controls; V<0.05 vs. OVX controls; ><0.05 vs. OVX +■ 1 mg. 

increases in weight of the liver (42%), lungs (19%), spleen (14%) and adrenal 
glands (39%) were seen when compared to those of sham controls. 

Effects in tibial length 

Figure 3 shows the changes in left tibial length: 37.7 + 0.92 mm at day (3 months 
of age) and 39.0 + 0.4 mm at day 90 (6 months of age), a significant increase with 
age. The tibial length in OVX rats treated with 1 and 6 mg PGE 2 /kg/day were 
longer compared to sham-OVX (39.7 + 0.56 mm at 1 mg and 39.4 + 0.31 mm at 6 
mg vs. 39.0 ±0.4 mm at sham-OVX). 

Qualitative observation of microradiographs 

The primary spongiosa and growth plate were thinner in sham controls than in 
basal controls (Figs. 4B vs. A). No obvious bone mass change was found in the 
secondary spongiosa between basal (3 months of age) and sham (6 months of age) 
controls. Thinner and more dense primary spongiosa and less bone mass 




Fig. 4. Microradiographs showing cancellous bone changes in proximal tibial metaphyses from basal (A), 
sham-operated (B), OVX (C) controls and OVX rats treated with 1 (D) and 6 (E) mg PGE 2 /kg/day. 
Thinner primary spongiosa and less bone mass throughout the whole metaphysis were observed in OVX 
control (C) than that in the sham control (B). Bone mass in OVX rat treated with 1 mg of PGE 2 (D) was 
greater than that in OVX control (C) and about equal to that in sham control (B). Bone mass in OVX rat 
treated with 6 mg of PGE 2 (E) was greater than that in both OVX (C) and sham (B) controls ( x 10.5). 



51 



throughout the entire metaphysis were observed in OVX controls than in sham 
controls (Figs. 4C vs. B). In the 1-mg PGEi/kg/day-treated OVX rat metaphysis, 
the primary spongiosa was wider, the bone mass was greater, and the trabeculae 
were thicker than in OVX controls (Figs. 4D vs. C), and equal to that in sham 
controls (Fig. 4B). The primary spongiosa width, bone mass and trabecular width 
in the 6-mg PGE 2 /kg day-treated OVX rat metaphysis (Fig. 4E) was much wider 
than that in any other group (Figs. 4A-D). 

Effects on bone mass and microanatomical structure 

Figures 5-7 and Table 1 show that trabecular bone area, width, number, separa- 
tion and microanatomical structural indices changed in aging, OVX controls and 
OVX rats treated with 1 and 6 mg PGE 2 /kg/day. 

Effects of aging. Between 3 and 6 months of age, there was no significant 
difference in trabecular bone area, width, number and separation, and all tissue- 



Trabecular bone area (%) 



B. Trabecular width (u.m) 
70 i 




C. Trabecular number (#/mm) 

5' 



D. Trabecular separation (jim) 



5000 -i 



4000 




I 



V, 



V 

T 



Fig. 5. Static histomorphometric indices of the proximal tibial metaphyseal trabeculae in basal, sham- 
operated and OVX controls and PGE2-treated OVX rats. Y error bar represents standard deviation. 
V<0.05 vs. basal controls; "'P<0.05 vs. sham-operated controls; */><0.05 vs. OVX controls; V<0.05 

vs. OVX + 1 mg. 



52 



based structural indices (Figs. 5 and 6, Table 1). However, when sham controls 
were compared to basal controls, the trabecular-bone-based density of node, 
node to node, and node to free end increased while cortex to free end and free 
end to free end decreased (Fig. 7). 



A. Node/TV (#/mm A 2) 




B. Node to node/TV (#/mm A 2) 

4.5- 




C. Cortex to node/TV (#/mm A 2) 

2.5' 




D. Cortex to free end/TV (#/mm«2) 

2.0" 



E. Free end to free end/TV (#/mm A 2) 

10 -I 





F. Node to free end/TV («/mm»2) 



8- 

7" 


-- 


6" 

5- 


T 


4- 






3" 






2" 






1 - 

n- 




-3 § 




Fig. 6. Tissue-area-based microanatomical structural indices of [he proximal tibial metaphyseal trabeculae 
in basal, sham-operated, OVX controls and PGE 2 -treated OVX rats. Y error bar represents standard 
deviation. t /'<0.05 vs. basal controls; ®/ > <0.05 vs. sham-operated controls; */ > <0.05 vs. OVX controls; 

"/><0.05 vs. OVX + 1 mg. 



53 



Effects ofOVX. Compared to sham-operated controls, OVX resulted in: (1) an 
80% decrease in percent trabecular bone area, and a 77% decrease in trabecular 
number; (2) 960% increase in trabecular separation (Fig. 5); (3) a decrease in all 
tissue-based microanatomical structural indices (from 60% to 97%, Fig. 6); (4) a 



A. Node/BV (#/mm A 2) 
30- 

t 




B. Node to node/BV (#/mm A 2) 
20- 




C. Cortex to node/BV (#/mm A 2) 
io- 




D. Cortex to free end/BV (#/mm A 2) 

40" 




E. Free end to free end/BV (#/mm A 2) 

140 "I 




F. Node to free end/BV (#/mm'2) 

50- 

t # 

40 



30- 



20 



10 











Fig. 7. Trabecular-bone-area-based microanatomical structural indices of proximal tibial metaphyseal 
trabeculae in basal, sham-operated, OVX controls and PGE2-treated OVX rats. Y error bar represents 
standard deviation. f /><0.05 vs. basal controls: ®P<0.05 vs. sham-operated controls; *<0.05 vs. OVX 

controls; 'P<0.05 vs. OVX+ 1 mg. 



54 

decrease in trabecular-bone-based density of node (85%), node to node (89%), 
node to free end (90%), and an increase in the indices for free end to free end 
(102%) and cortex to free end (282%) (Fig. 7). The ratio of node to free end 
decreased to 13% of those of basal and sham controls (Table 1). 

Effects ofPGE; on O VX rats (compared to O VX controls). In PGE 2 -treated OVX 
rats, trabecular bone area, width and number increased and trabecular 
separation decreased. Trabecular bone area increased 343 and 626% over 
OVX controls for 1- and 6-mg PGE 2 /kg, day-treated OVX rats. Similarly 
trabecular width increased 35% for the 6-mg dose, and trabecular number 
increased 233% and 359% for the 1- and 6-mg doses. Trabecular separation 
decreased 87% and 91% for 1- and 6-mg PGE 2 /kg/day-treated OVX rats (Fig. 
5). In 1- and 6-mg PGE 2 /kg/day-treated OVX rats, all tissue-based structural 
indices increased from 79 to 2187% (Fig. 6). Trabecular-bone-based density of 
node, node to node and node to free end were elevated while free end to free end 
and cortex to free end declined in 1- and 6-mg PGE 2 /kg/day-treated OVX rats 
(Fig. 7). Further, there were 6- and 10-fold increases in node to free end ratios 
after 1 and 6 mg PGE 2 treatment (Table 1). 

Effects of PGE : on OVX rats (compared to sham controls). In PGE 2 -treated OVX 
rats, trabecular bone area in 1-mg PGE 2 /kg/day-treated OVX rats was 
unchanged (Fig. 5A), but trabecular number declined by 21% and trabecular 
width was elevated by 23% (Figs. 5B and C). At the 6-mg dose level, percent 
trabecular bone area increased by 61% and trabecular width by 44% (Fig. 5). In 
1-mg PGE 2 /kg/day-treated OVX rats, the tissue-based density of cortex to node, 
free end to free end and node to free end were lower than sham controls (22 to 
37%) (Fig. 6). None of the trabecular bone-based microanatomical structure 
indices differed from sham controls at both 1- and 6-mg dose levels (Fig. 7). 

Effects on longitudinal bone growth and growth plate thickness 
When compared to the rate of longitudinal growth at 3 months of age, the 
longitudinal growth rate at 6 months of age had decreased by 71%. However 
OVX controls showed a 21% increase in longitudinal growth rate over sham- 
Table 1 
Ratio of node to free end of proximal tibial trabeculae a 

Ratio vs. basal vs. sham vs. OVX vs. OVX + 1 mg 

Basal controls 0.23 ±0.04 

Sham controls 0.23±0.08 -3% (ns) 

OVX controls 0.03±0.05 —87% (/ > <0.001) -87% (/><0.00l) 

OVX + 1 mg 0.20 + 0.05 -15% (ns) -13% (ns) +578% (/ > <0.05) 

OVX + 6mg 0.34±0.22 +45% (ns) +50% (ns) + 1054% (V<0.05) +72% (ns) 



a Calculated as: node no. /(free end to free end no. + cortex to free end no. + node to free end no), 
ns: nonsignificant difference. 



55 



OVX controls (Fig. 8A). In the 6-mg PGE 2 -treated OVX rats, longitudinal 
growth rate increased compared to sham (48%) and OVX (22%) controls (Fig. 
8A). Growth plate thickness decreased in sham controls compared to basal 
controls, while it increased in ovariectomized rats compared to sham controls 
(33%). During the experimental periods, there was a 5.8 + 0. 1 -mm new metaphy- 
sis generated (mean accumulated longitudinal growth) in sham-OVX control rats, 
while there was a metaphysis of 6.1 ±0.3, 6.3 ±0.2 and 6.5 ±0.2 mm in OVX 
controls, 1- and 6-mg PGE 2 -treated OVX rats, respectively (Fig. 8C): an increase 
in the latter three groups from the sham controls (Fig. 8C). 

Effects on dynamic histomorphometry 

Effects of aging. There were significant decreases in labeled perimeter (33%), 
bone- and tissue-area-based bone formation rates (38 and 46%, respectively), 
and significant increases in the formation (49%) and resorption (1 12%) periods 
in sham-OVX controls (6 months of age) compared to basal controls (3 months 
of age) (Figs. 9B, D and E, 10B and C). 



A. 


Longitu 


aina 


1 g 


row 


th 


55- 




T 






45" 










35- 
















@ 


@ 


25- 






t 


t 


15- 






t 

T- 




9 w 






< - 




1 

a 


I 


1 


go 



?W9 



B. Growth plate thickness ((im) 
no - 




C. Newly metaphysis generated (mm) 

7.0 1 




Fig. 8. Longitudinal growth rate, growth plate thickness and newly generated metaphysis of proximal 

tibial metaphyses in basal, sham-operated, OVX controls and PGEwreated OVX rats. Y error bar 

represents standard deviation. *P<0.05 vs. basal controls; ^PkO.OS vs. sham-operated controls; 

*P<0.05 vs. OVX controls; 'P<0.05 vs. OVX ■*■ 1 mg. 



56 



Effects of ovariectomy. There were increases in percent osteoid perimeter 
(167%), labeled perimeter (109%), bone formation rate-bone area referent 
(126%), eroded perimeter (80%) and activation frequency (245%), and 



A. Osteoid perimeter ( 

45 -i 



Labeling perimeter (%) 




C. Mineral apposition rate (|im/d) 
2.0 



D. Bone formation rate/BV (%/y) 

600- 




E. Bone formation rate/TV (%/y) 
120" 



F. Eroded perimeter (%) 




25- 
20- 


t 


@ 


15" 
10- 


T 


T 


r 
/ 

/ 


\ 


5- 
n - 




1 

S3 


1 





Fig. 9. Dynamic histomorphometric indices of the proximal tibial metaphyseal trabeculae in basal, sham- 
operated. OVX controls and PGEi-treated OVX rats. Y error bar represents standard deviation. V<0.05 
vs. basal controls; ,a */ 3 <0.05 vs. sham-operated controls; *?<0.05 vs. OVX controls; ><0.05 vs. OVX + 

1 mg. 



57 



significant decreases in bone formation rate-tissue referent (51%), trabecular 
wall width (42%), formation period (39%), resorption period (70%), quiescent 
period (83%) and remodeling period (52%) in OVX controls compared to sham- 
OVX controls (Figs. 9 and 10). 



A. Trabecular wall width (|im) 

# 




B. Formation period (days) 
35- 




C. Resorption period (days) 



25 -\ 



20" 




D. Quiescent period (days) 
250 n 



200" 




150- 



100 



50" 



E. Remodeling period (days) 

70-1 




F. Activation frequency (#/year) 

12 1 




Fig. 10. Trabecular wall width, formation, resorption and quiescent periods and activation frequency of 

proximal tibial metaphyseal trabeculae in basal, sham-operated, OVX controls and PGE 2 -treated OVX 

rats. Y error bar represents standard deviation. V<0.05 vs. basal controls; ( */ , <0.05 vs. sham-operated 

controls; "P<0.0S vs. OVX controls; ><0.05 vs. OVX + 1 mg 



58 

Effects of PGE 2 on OVX rats (compared to OVX controls). There was a 
significant decrease in percent osteoid perimeter (27 and 28%), and a significant 
increase in mineral apposition rate (15 and 29%), bone formation rate-tissue 
area referent (291 and 652%) and wall width (146 and 170%) in 1- and 6-mg 
PGE 2 -treated OVX rats (Figs. 9 and 10A). Bone formation, resorption, 
quiescent and remodeling periods were shortened (from 79 to 244%), and the 
index of activation frequency of bone remodeling decreased (71 and 53%) in 
PGE 2 -treated OVX rats (Figs. 10B-F). 

Effects of PGE : on OVX rats (compared to sham-OVX controls). Except for 
mineral apposition rate in 1-mg PGE 2 -treated OVX rats, all bone formation 
parameters were either increased significantly (42 to 270%) or, in the case of the 
bone resorption parameter, was not changed in the 1- and 6-mg PGE 2 /kg/day- 
treated OVX rats (Figs. 9A-F). Wall width was thicker by 56% in 6-mg PGE 2 - 
treated OVX rats (Fig. 10A). Bone formation, resorption, quiescent and 
remodeling periods, and activation frequency were nonsignificantly changed in 
PGE 2 -treated OVX rats (Figs. 10B-F). Furthermore, diffuse-labeled new bone, 
as an index of woven bone formation, was seen in all 1- and 6-mg PGE 2 /kg/day 
treated rats (0.01 1 ±0.006 mm 2 at 1 mg and 0.022 + 0.004 mm 2 at 6 mg, 
respectively) and was not seen in any basal, sham-OVX or OVX controls. 



Discussion 

Some interesting cancellous bone data were generated between sham-operated 
controls (6 months of age) and basal controls (3 months of age). Between 3 and 6 
months of age, the trabecular bone area, width and number was nonsignificantly 
decreased in proximal tibial metaphyses. But the dynamic parameters show that 
trabecular labeling perimeter, bone formation rates (bone area and tissue area 
referent) and longitudinal growth rates declined significantly, while formation 
and resorption periods were elevated significantly, and trabecular eroded peri- 
meter was not significantly different. Taken all together, these changes indicate 
that aging decreases bone formation, prolongs bone remodeling, but does not 
change bone resorption. The age-related changes in the indices of trabecular 
microanatomical structure indicate that the nonsignificant decrease in trabecular 
bone area between these ages coincides with the thinning of trabeculae and the 
loss of some disconnected trabeculae (CTF and FTF). At the same time there was 
no loss of connected trabeculae (NTN). The net effect was that the connections 
per unit bone area (node/BV and NTN/BV) were improved. Possibly, the 
explanation is that the mechanical loading on disconnected trabeculae is less 
than that in connected trabeculae, so that the disconnected trabeculae will be the 
first to go in age-related bone loss. 

The ovariectomy-induced bone loss rat model is widely accepted for studying 
the prevention and treatment of postmenopausal osteoporosis [39^*0]. In young- 
er rats, ovariectomy results in a dramatic decrease in cancellous bone mass 



59 

associated with an increase in bone turnover with bone resorption exceeding 
formation [38-46]. Our findings of a decrease in trabecular bone area, the 
increment of percent eroded perimeter, percent osteoid and labeling perimeter, 
bone formation rate-bone area referent, and the decrement of formation, resorp- 
tion, remodeling and quiescent periods in OVX control rats are consistent with 
findings reported previously [38-46]. Mineral apposition rate was increased 
significantly in OVX rats in other reports [23,44,47], but this change was not 
seen in the current study. Wronski et al. reported that longitudinal growth in 
proximal tibial metaphyses increased significantly in OVX rats 14 and 35 days 
post ovariectomy and returned to control levels thereafter [23,38,42-44]. How- 
ever, we still observed a significantly increased longitudinal growth rate at 90 days 
post ovariectomy. The activation frequency index was elevated in our OVX rats 
which further confirms the increment in bone turnover. Even though bone 
formation per unit bone area increased in OVX rats, bone formation rate-tissue 
area referent declined in OVX rats, indicating the absolute bone-forming area 
decreased in these rats. 

We also detailed the changes in microanatomical structural indices of trabe- 
cular bone in proximal tibial metaphyses in OVX rats in the current paper, data 
lacking in previous reports [38-46]. These indices showed that the OVX-induced 
bone loss is accompanied by decreased interconnectedness of normal trabecular 
structural pattern. The decreased interconnectedness of trabeculae was most 
obvious when one compares the ratio of node to free end between OVX and 
basal and sham-OVX control rats (Table 1). 

The findings from the present study demonstrate that bone loss due to ovarian 
hormone deficiency can be prevented by a low-dose (1 mg) daily administration of 
PGE 2 . Furthermore a higher-dose (6 mg) daily administration of PGE 2 not only 
prevents bone loss but also adds extra bone to the proximal tibial metaphyses. 
PGE 2 at the 1-mg dose level significantly increased trabecular bone area, trabe- 
cular width, trabecular node density, density of node to node, ratio of node to free 
end, and thus significantly decreased trabecular separation from OVX controls. 
At this dose level, these same parameters did not differ significantly from sham- 
operated controls. However, at the 6-mg dose level PGE 2 , there were significant 
increases in trabecular bone area, trabecular width, trabecular node density, 
density of node to node, ratio of node to free end, while there was a significant 
decrease in trabecular separation from both OVX and sham-operated controls. 
The changes in indices of trabecular bone microanatomical structure indicated 
that PGE 2 prevented bone loss as well as the disconnection of existing trabeculae 
(Table 1). 

Prevention of bone loss after ovariectomy by many agents, such as estrogen, 
parathyroid hormone, bisphosphonate have been studied [39,40,47-58]. Estrogen 
prevents bone loss induced by ovariectomy by inhibiting bone turnover and bone 
resorption [39,40,47-50]. Bone loss after ovariectomy can be prevented and 
restored by human parathyroid hormone by retarding bone resorption and 
enhancing bone formation [52-57]. Bisphosphonate decreases both bone forma- 
tion and resorption in ovariectomized rats so as to prevent bone loss [58]. Unlike 



60 

the other agents [39,40,47-58], PGE 2 given to OVX rats does not inhibit the 
increment in percent eroded perimeter induced by ovariectomy, instead it stimu- 
lates lamellar bone formation, activates woven bone formation and creates a 
positive bone balance. Surprisingly, PGE 2 -treated OVX rats had lower bone 
turnover rate than the OVX controls did (decrease in activation frequency 
index, and increase in formation, resorption and quiescent periods), since we 
previously reported that when PGE 2 was given to intact rats for 30 and 60 days, it 
increased bone turnover in favor of bone formation compared to age-related 
controls [16-22]. 

Finally, we conclude that PGE 2 can prevent bone loss due to estrogen 
deficiency (ovariectomy) and add extra cancellous bone to these ovariectomized 
rats by stimulating bone formation exceeding resorption, and activating woven 
bone formation. Our findings support the strategy of the use of bone stimulation 
agents in place of anti-resorptive agents in the prevention of estrogen depletion 
bone loss (postmenopausal osteoporosis). 



Acknowledgment 

This work was supported mainly by a grant from the National Institutes of 
Health (AR-38346). It was also partially supported by a research contract and 
grant from the Department of Energy (DE-AC02-76EV001 19 and DE-FG02- 
89ER60764), and a grant from the National Aeronautics and Space Administra- 
tion (NAG-2-435). We thank Dr. Charles Hall and Mr. Ronald E. Lane of the 
Upjohn Company for the PGE 2 . We also thank R.B. Setterberg and Drs. Q.Q. 
Zeng, L.Y. Tang and B.Y. Lin for their expert assistance and advice. 



References 

1 Lund JE, Brown WP, Tregerman L. The toxicology of PGE,. In: Wu KK, Rossi ED, eds. 
Prostaglandins in clinical medicine: Cardiovascular and thrombotic disorders, Year Book. Chicago IL: 
Medical Pub. Inc. 1982; 93-103. 

2 Ueno K, Haba T, Woodbury D, Price P, Anderson R. Jee WSS. The effects of prostaglandin E 2 in 
rapidly growing rats: Depressed longitudinal and radial growth and increased metaphyseal hard tissue 
mass. Bone 1985;6:79-86. 

3 Furuta Y, Jee WSS. Effect of 16,16-dimethyl prostaglandin E 2 methyl ester on weanling rat skeleton: 
Daily and systemic administration. Anat Rec 1986:215:305-316. 

4 High WB. Prostaglandins and bone remodeling. Calcif Tissue Int 1987;41:295-296. 

5 High WB. Effects of orally administered prostaglandin E 2 on cortical bone turnover in adult dogs: A 
histomorphometric study. Bone 1988;8:363-373. 

6 Shih M-S, Norrdin RW. Effect of prostaglandin E| on regional haversian remodeling in beagles with 
fractured ribs: A histomorphometric study. Bone 1987;8:87-90. 

7 Shih M-S. Norrdin RW. Effect of prostaglandin E 2 on regional cortico-endosteal remodeling in 
beagles with fractured ribs: A histomorphometric study. Bone Miner 1987;3:27-34. 

8 Norrdin RW, Shih M-S. Systemic effects of prostaglandin E 2 on vertebral trabecular remodeling in 
beagles used in healing study. Calcif Tissue Int 1988;42:363-368. 

9 Jee WSS, Ueno K, Deng YP, Woodbury DM. The effects of prostaglandin E 2 in rapidly growing rats: 



61 

Increased metaphyseal hard tissue and cortico-endosteal bone formation. Calcif Tissue Int 
1985;37:148-157. 

10 Jee WSS, Ueno K, Kimmel DB, Woodbury DM, Price P, Woodbury LA. The role of bone cells in 
increasing metaphyseal hard tissue in rapidly growing rats treated with prostaglandin E 2 . Bone 
1987;8:171-178. 

1 1 Li XJ, Jee WSS, Li LY, Patterson-Buckendahl P. Transient effects of subcutaneously administered 
prostaglandin E 2 on cancellous and cortical bone in young adult dogs. Bone 1990;11:353-364. 

12 Marks SC Jr, Miller SC. Local infusion of prostaglandin E| stimulates mandibular bone formation in 
vivo. J Oral Pathol 1988;17:500-505. 

13 Sone K, Tashiro M, Fujinaga T, Romomasu T, Tokayama K, Kurome T. Long-term low dose 
prostaglandin E, administration. J Pediatr 1980;97:834-836. 

14 Ueda K, Saito A, Nakano H, Aoshima M, Yokota M, Muraoka R, Iwaya T. Cortical hyperostosis 
following long-term administration of prostaglandin E 2 in infants with cyanotic congenital heart 
disease. J Pediatr 1980;97:834-836. 

15 Ringel RW, Brenner JI, Henry PH, Burns JE, Moulton AL, Berman MA. Prostaglandin induced 
periostitis: A complication of long-term PGEj infusion in an infant with congenital heart disease. 
Radiology 1982;142:657-658. 

16 Mori S. Jee WSS, Li XJ. Production of new trabecular bone in osteopenic ovariectomized rats by 
prostaglandin E 2 . Calcif Tissue Int 1992;50:80-87. 

17 Mori S, Jee WSS, Li XJ, Chan S, Kimmel DB. Effects of prostaglandin E 2 on production of new 
cancellous bone in axial skeleton of ovariectomized rats. Bone 1991;11:103-113. 

18 Jee WSS, Mori S, Li XJ, Chan S. Prostaglandin E 2 enhances cortical bone mass and activates 
intracortical bone remodeling in intact and ovariectomized female rats. Bone 1991;11:253-266. 

19 Ke HZ, Jee WSS, Li XJ, Mori S, Kimmel DB. Effect of long term daily administration of 
prostaglandin E 2 on maintaining elevated proximal tibial metaphyseal cancellous bone in adult male 
rats. Calcif Tissue Int 1992;50:245-252. 

20 Ke HZ. Li XJ, Jee WSS. Partial loss of anabolic effects of prostaglandin E 2 on bone after its 
withdrawal in rats. Bone 1991;12:173-183. 

21 Ke HZ, Jee WSS. The effects of daily administration of prostaglandin E 2 and its withdrawal on the 
lumbar vertebral bodies in male rats. Anat Rec (in press). 

22 Jee WSS, Ke HZ, Li XJ. Long-term anabolic effect of prostaglandin E 2 on tibial diaphysel bone in 
male rats. Bone Miner 1991;15:33-55. 

23 Wronski TJ, Dann LM, Scott KS, Cintron M. Long-term effects of ovariectomy and aging on the rat 
skeleton. Calcif Tissue Int 1989;45:360-366. 

24 Baron R, Tross R, Vignery A. Evidence of sequential remodeling in rat trabecular bone: Morphology, 
dynamic histomorphometry and changes during skeletal maturation. Anat Rec 1984;208:137-145. 

25 Akamine T, Jee WSS, Ke HZ, Mori S, Akamine T. Prostaglandin E 2 prevents bone loss and adds extra 
bone to immobilized distal femoral metaphysis in female rats. Bone 1992; 13:1 1-22. 

26 Katz IA, Jee WSS, Joffe II, Stein B, Takizawa M, Jacobs TW, Setterberg R, Lin BY, Tang LY, Ke 
HZ, Zeng QQ, Berlin J, Epstein S. Prostaglandin E 2 alleviates cyclosporin A-induced bone loss in the 
rat. J Bone Miner Res (in press). 

27 Li XJ, Jee WSS, Ke HZ, Mori S, Akamine T. Age-related changes of cancellous and cortical bone 
histomorphometry in female Sprague-Dawley rats. Cells Mater 1991;Suppl, 1 ;25— 35. 

28 Jee WSS, Inoue J, Jee KW, Haba T. Histomorphometric assay of the growing long bone. In: 
Takahashi, H., ed. Handbook of bone morphology. Niigata City, Japan: Nishimusa, 1983; 101-122. 

29 Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM. Recker RR. 
Bone histomorphometry: Standardization of nomenclature, symbols, and units. J Bone Miner Res 
1987;2:595-610. 

30 Kimmel DB, Jee WSS. A quantitative histologic analysis of the growing long bone metaphysis. Calcif 
Tissue Int 1980;32:113-122. 

31 Garrahan NJ, Mellish RWE, Compston JE. A new method for the two-dimensional analysis of bone 
structure in human iliac crest biopsies. J Microsc 1986;143:341-349. 

32 Compston JE, Mellish RWE, Garrahan NJ. Age-related changes in iliac crest trabecular 
microanatomic bone structure in man. Bone 1987;8:289-292. 



62 

33 Compston JE, Mellish RWE, Croucher P. Newcombe R. Garrahan NJ. Structural mechanisms of 
trabecular bone loss in man. Bone Miner 1989;6:339-350. 

34 Compston JE. Croucher PI. Histomorphometric assessment of trabecular bone remodeling in 
osteoporosis. Bone Miner 1991;14:91-102. 

35 Frost HM. Bone histomorphometry: Analysis of trabecular bone dynamics. In: Recker RR, ed. Bone 
histomorphometry: Techniques and interpretation. Boca Raton. FL: CRC Press. 1983; 1 09—1 3 1 . 

36 Jee WSS. Introduction to skeletal function: Structural and metabolic aspects In: Bronner F, Worell 
RV, eds. A basic science primer in orthopaedics. Baltimore: Williams and Wilkins. 1991; 3-34. 

37 Neter J, Wasserman W, Whitmore GA. Applied statistics. Boston: Allyn and Bacon, 1982:544-572. 

38 Wronski TJ. Schenck PA, Cintron M, Walsh CC. Effect of body weight on osteopenia in 
ovariectomized rats. Calcif Tissue Int 1987;40:155-159. 

39 Kalu DN. The ovariectomized rat model of postmenopausal bone loss. Bone Miner 1991;15:175-192. 

40 Wronski TJ, Yen C-F. The ovariectomized rat as an animal model for postmenopausal bone loss. Cells 
Mater 1991; Suppl 1:69-74. 

41 Miller SC, Bowman BM, Miller MA, Bagi CM. Calcium absorption and osseous organ-, tissue-, and 
envelope-specific changes following ovariectomy in rats. Bone 1991;12:439-446. 

42 Wronski TJ. Walsh CC, Ignaszewski LA. Histological evidence for osteopenia and increased bone 
turnover in ovariectomized rats. Bone 1986:7:119-123. 

43 Wronski TJ. Lowry PL. Walsh CC. Ignaszewski LA. Skeletal alteration in ovariectomized rats. Calcif 
Tissue Int 1985:37:324-328. 

44 Wronski TJ, Cintron M, Dann LM. Temporal relationship between bone loss and increased bone 
turnover in ovariectomized rats. Calcif Tissue Int 1988:43:179-183. 

45 Wronski TJ. Dann LM, Horner SL. Time course of vertebral osteopenia in ovariectomized rats. Bone 
1989:10:295-301. 

46 Kalu DN, Liu C-C, Hardin RR, Hollis BW The aged rat model of ovarian hormone deficiency bone 
loss. Endocrinology 1989;24:7-16. 

47 Kalu DN, Liu C-C, Salerno E, Hollis B, Echon R. Ray M. Skeletal response of ovariectomized rats to 
low and high dose of 17/J-estradiol. Bone Miner 1991;14:175-187. 

48 Wronski TJ, Cintron M, Doherty AL, Dann LM. Estrogen treatment prevents osteopenia and 
depressed bone turnover in ovariectomized rats. Endocrinology 1988;123:681-686. 

49 Wronski TJ, Yen C-F. Scott KS. Estrogen and diphosphonate treatment provide long-term protection 
against osteopenia in ovariectomized rats. J Bone Miner Res 1991;6:387-394. 

50 Turner RT, Vandersteenhoven J A, Bell NH. The effects of ovariectomy and 17/?-estradiol on cortical 
bone histomorphometry in growing rats. J Bone Miner Res 1987;2:115-122. 

51 Gunness-Hey M, Hock JM. Loss of anabolic effect of parathyroid hormone on bone after 
discontinuation of hormone in rats. Bone 1989;10:447-452. 

52 Hock JM, Gera I, Fonseca J, Raisy LG. Human parathyroid hormone-(l-34) increases bone mass in 
ovariectomized and orchidectomized rats. Endocrinology 1988:122:2899-2904. 

53 Holtrop ME, Raisz LG. Comparison of the effects of 1,25-dihydroxycholecalciferol, prostaglandin E 2 , 
and osteoclast-activating factor with parathyroid hormone on the ultrastructure of osteoclasts in 
cultured long bones of fetal rats. Calcif Tissue Int 1979;29:201-205. 

54 Liu C-C, Kalu DN. Human parathyroid hormone-(l -34) prevents bone loss and augments bone 
formation in sexually mature ovariectomized rats. J Bone Miner Res 1990;5:973-982. 

55 Liu C-C, Kalu DN, Salerno E, Echon R, Hollis BW, Ray M. Preexisting bone loss associated with 
ovariectomy in rats is reversed by parathyroid hormone. J Bone Miner Res 1991;10:1071-1080. 

56 Jee WSS, ed. The aged rat model for bone biology studies. Cells Mater 1991; Suppl 1:1-192. 

57 Hock JM, Wood RJ. Bone response to parathyroid hormone in aged rats. Cells Mater 1991; Suppl 
1:53-58. 

58 Seedor JG, Quartuccio HA, Thompson DD. The bisphosphonate alendronate (MK-217) inhibits bone 
loss due to ovariectomy in rats. J Bone Miner Res 1991;6:339-346.